Comparative Study of the Action of Bovine Duodenal Proteinases (Duodenases) on Polypeptide Substrates

E. A. Sokolova^1*, O. A. Mirgorodskaya², P. Roepstorff³, N. V. Savelyeva², and T. S. Zamolodchikova¹

¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117871 Russia; fax: (095) 310-7007; E-mail: elena@enzyme.siobc.ras.ru

²Institute of Cytology, Russian Academy of Sciences, Tikhoretskaya nab. 4, St. Petersburg, 194064 Russia; fax: (812) 247-0341

³Department of Molecular Biology, Odense University, DK-5230, Odense M, Denmark

^* To whom correspondence should be addressed.

Received March 17, 2000; Revision received August 22, 2000

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A comparative study of substrate specificity of bovine duodenal proteinases--chymotrypsin-like duodenase (ChlD) and dual-specificity duodenase (dsD)--was carried out using oligopeptide substrates (human proinsulin, glucagon, melittin, angiotensinogen fragment 1-14). ChlD displayed mainly chymotrypsin-like properties towards these substrates, hydrolyzing peptide bonds carboxy-terminally to bulky aliphatic or aromatic residues. In melittin, ChlD additionally cleaved peptide bonds after Thr and Ser residues. Dual-specificity duodenase (dsD) significantly restricted its specificity to only trypsin-like or only chymotrypsin-like or displayed full activity, combining both specificities, depending on substrate. Both ChlD and dsD efficiently hydrolyzed a single peptide bond (Phe8-His9) in angiotensinogen fragment 1-14. The kinetic parameters of angiotensinogen fragment 1-14 cleavage by ChlD and dsD were determined (k_cat/K_m = 80,500 M^-1·sec^-1 for ChlD and 103,000 M^-1·sec^-1 for dsD).

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KEY WORDS: duodenases, serine proteases, substrate specificity

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